Anti-inflammatory effect of HGF responses to traumatic oral ulcers using an HGF-Tg mouse model

Background: Hepatocyte growth factor (HGF) has been implicated in inhibiting diverse types of inflammation and promoting wound healing. In this study, we demonstrate the anti-inflammatory effect of HGF on traumatic ulcer of oral mucosa using HGF overexpression transgenic C57BL/6 (HGF-Tg) mice. In total, 29 C57BL/6 (WT) and 29 HGF-Tg mice were used in this study. Traumatic ulcer of left mucosa was performed by abrasion using a n 15 surgery knife. Ulcer tissues and serum samples were collected on 5 th day, histological score, expression of inflammatory cells and serum cytokines expression were measured and analyzed. Results: There existed statistical significant difference in connective Ly6G positive cells and NF-кB positive expression, HGF-Tg mice showed lower number of positive cells ( p =0.048, p =0.020 ). Eotaxin ( t -test, p =0.002), MIP-1gamma ( t -test, p =0.011), BLC ( t -test, p =0.03),Eotaxin-2 ( t -test, p =0.027), RANTES ( t -test, p =0.038), Lix ( t -test, p =0.04) and IL-3 ( t -test, p =0.047) had statistical significance higher expression in HGF-Tg mice. No significant difference of blood T cells ( p =0.998), Neutrophils ( p =0.331),Macrophages ( p =0.470) and CD4+/CD8+ ratio ( p =0.451) between HGF-Tg and WT group. Conclusions: We observed that HGF-Tg animals presented less Ly6G-positive neutrophil infiltration, higher levels of circulating cytokines and less NF- к B expression in connective tissue, with a positive effect on the healing of oral traumatic ulcers.


Background
Ulceration of the oral mucosa are relatively frequent and feature discontinuity of the epithelium [1,2].
They may be induced by many underlying etiologic factors including trauma, autoimmune/immunemediated disorder, infection, causing tissue broken and ulceration [3]. Clinically, these ulcers are usually can be painful and may affect chewing, swallowing speaking and dramatically affect the patient's life quality. Healing happens in spontaneous manner that may vary from 1 to 2 weeks, however, medication intervention is needed in case of chronic or severe ulcer. Antiseptic mouthwashes, steroids, alternate immunosuppressant medications and protective films/gels were recommended clinically to reduce pain and promote healing of oral ulcer [2,4]. Recent years, regenerative medicine concept is developed for an alternative to promote tissue repair with the understanding of the biological mechanism of wound healing. Application of growth factors has been investigated to promote ulcer healing [5]. Hepatocyte growth factor (HGF) is one of the growth factors that is considered as a new potential clinical drug in tissue healing [6].
HGF expression was found to be upregulated following a variety of injuries; it was postulated that cells of the immune system contribute to regulating the expression of HGF and c-Met following injury and inflammation [7,8,9].HGF promotes the development of immune cells and contributes to immune responses and tissue repair [10,11]. Moreover, HGF was reported to inhibit the secretion of inflammatory cytokines and to promote secretion of anti-inflammatory cytokines [12,13]. HGF was more recently reported to modulate the inflammatory environment and enhance wound healing and regeneration in animal models of inflammation-associated diseases in skin inflammation models [14], multiple sclerosis models [15] and models [16].
HGF can mediate skin wound healing by accelerating regeneration of epithelial cells [17] and by maintaining normal immune function [14]. HGF also regulates desirable wound healing by mediating oral mucosal fibroblast function [18]. Accordingly, we hypothesized that using HGF will promote oral ulcer healing and remodeling. Because the short biological half life of internal HGF protein, intravenous medicine delivery can not ensure the effective function of HGF in the traumatic tissues, we established HGF-overexpressing transgenic (HGF-Tg) mice whose HGF expression level is higher than in wild type (WT) mice [19]. Our previous study found that HGF exerted anti-inflammatory effects by reducing both epithelial apoptosis and connective tissue TNFα expression in oral traumatic ulcers [19]. In this study, we tested the effects of HGF on inflammatory cells and cytokine expression in healing from traumatic injury of oral mucosa using HGF-Tg mice. (HGF-Tg) mice were used in this study [19]. HGF is high expressed in oral mucosa of HGF-Tg mice( Fig   1). In total, 29 WT and 29 HGF-Tg mice aged 8-10 weeks (22.8 g±5.2 g, 8-10 weeks) were used in this experiment. Mice were kept under 12-hour/12-hour light/dark cycles, with free access to food and water, in the Central Animal Laboratory of Guangzhou Medical University.

Oral mucosa traumatic ulcer protocol
Oral mucosa traumatic ulcers were generated using the method described by Cavalcante in 2011 [4].
Briefly, the mice were anesthetized with 0.1-0.2 mlof pentobarbital sodium (0.1%) via intraperitoneal injection, and the oral mucosa was sterilized using 0.12% chlorhexidine gluconate. The ulceration was performed on the left oral mucosa using a number 15 scalpel blade; a 5-mm marker was used for standardization. The protocol was performed by the same investigator (Guanying Chen) for standardization. We choose 5th day as the sacrifice day because the mice lose weight during the 5 first days [4].

Blood and tissue sample collection
The mice were weighed on day zero and on the day of sacrifice (the 5 th day), Weight loss=(final weight-initial weight). The mice were sacrificed by ether anesthesia and neck dislocation. Blood samples were collected and centrifuged for 10 min at 200 x g, after which the separated serum was collected and stored at -80 °C. The blood cells were collected for flow cytometry analysis. The mucosa containing the ulcers were collected and fixed in 10% formalin. After being embedded in paraffin, the tissue was sectioned into 2-µm slices that were then mounted on slides and stained with hematoxylin and eosin (HE) for inflammation score evaluation (from 0 to 4 according to published evaluation criteria) [2].Weighing, blood collection and sample collection were performed separately on the 5 th day. Carbonne, France) at a wavelength of 532 nm and a resolution of 10 µm. All results were analyzed using Q-Analyzer software for QAM-cyt-5. Healthy wild-type mice (n=3), WT mice with ulcers (n=5) and HGF-Tg mice with ulcers (n=8) were included in this analysis.

Flow cytometry analysis
Immune cells from WT (n=29) and HGF-Tg mice (n=29) blood were isolated after red blood cell lysis.

Statistical analysis
SPSS 19.0 (IBM, USA) was used to calculate statistical significance between groups. The unpaired ttest and ANOVA were used to compare the differences between groups. Quantitative data are expressed as the mean ± standard error (mean±SE). GraphPad Prism 6 software (La Jolla, CA, USA) was used to plot the comparison of means, and p≤0.05 was considered a statistically significant difference.

Weight loss and inflammation score analysis
All mice were alive on the sacrifice day, HGF-Tg mice (0.67±1.38 g) had significantly less weight loss compared with WT mice (-0.07±1.94 g) (t-test, p=0.038); Microscopic analysis of the cheek trauma of HGF-Tg mice on the 5 th day revealed the presence of less ulceration associated with moderate fibrosis and chronic inflammatory processes. In WT ulcer mice, we found a persistence of ulcers and mixed inflammatory infiltrates (neutrophils and mononuclear cells). HGF-Tg group (mean±SE: 2.55±0.67) exhibited less ulceration and inflammation than did WT group (mean±SE: 3.32±0.65) (t-test, p=0.000, Fig 2A).

RayBiotech (AAM-INF-G1-8) antibody array experiment
In this assay, compared with WT group, we detected 38 greater and 2 lesser (MIP-1 alpha, Fractalkine) amounts of production of serum cytokines in HGF-Tg group (Table 1). In terms of higher serum expression, we found 1-13.5-fold higher expression in HGF-Tg group. Eotaxin (t-test, p=0.002), p=0.037) and IL-3 showed significantly higher expression levels than did control group. IL-6 (t-test, p=0.050) was significantly lower and TCA-3 (t-test, p=0.034) was significantly higher in WT group than in control group (Fig 3A and 3B).

Flow cytometry analysis
To characterize the impact of oral mucosa ulcer on circulating inflammatory cells, we performed flow cytometry analysis 5 days after oral traumatic ulcer induction. T cells were gated by CD45+/ CD8+ or CD4+; neutrophils were gated by CD45+/ CD11b+/Ly6G+; and macrophages were gated by CD45+/ Ly6G-/ CD11b+/ F4/80+. We found that there were no significant differences in terms of blood T cells (p=0.998), neutrophils (p=0.331) and macrophages (p=0.470) between HGF-Tg and WT mice. Flow cytometry revealed that there were no significant differences in the CD4+/CD8+ ratio between the groups (p=0.451) (Fig. 4).

Discussion
The healing process of wound is a complex, programmed events of genetic and biological series.
However, non-healing and chronic ulcer are often more challenging to clinical prognosis and public health [21]. Recent years, some reports illustrated the potential clinical usage of growth factors in wound healing and tissue repair. HGF is one of the potential growth factors which participate in the whole process of wound healing [6,22]. While the function of HGF in cancer development and epithelial cell activities has been well discussed, the role of HGF signaling in inflammation and wound healing is gaining increasing focus. High HGF expression has been shown to mediate antiinflammatory action and regeration in a variety of inflammatory and traumatic models [13, 23,24,25].
The aim of this study was to evaluate anti-inflammatory activity of HGF on an oral mucosa traumatic ulcer model in rats, an appropriate model for clinical oral ulcer [4].
Recent studies have described the indispensable contribution of HGF to wounding healing by inhibiting immune cell infiltration and inflammation in tissues [8,26]. We found that HGF-Tg mice presented less weight loss, less epithelial ulceration and less infiltration of Ly6G+ neutrophils as shown by histological and immunohistochemical observation on day 5. Neutrophils are the major infiltrating cells in wounds, especially in the early phase [27], our finding indicates that the effect of HGF in the healing of oral wounds involves decreasing the number of neutrophils in connective tissue of wound sites rather than the number of circulating immune cells; the mechanism of this phenomenon merits deep study. One study showed that HGF-specific c-Met knockout mice showed significantly severe neutrophil infiltration in non-alcoholic fatty liver disease (NAFLD) [28]. HGF treatment can decrease the Lymphocytes of the spleen in mice model [16]. Overexpression of HGF was tested as a way to enhance connective inflammation repair rather than re-epithelialization of wounds [22,29]. In terms of the effect of HGF on circulating immune cells and CD45+ T cells in connective tissue of oral ulcers, HGF failed to induce a significant decrease in circulating immune cell numbers or tissue T cells compared with WT mice.
Inflammation cytokines mediate complex functions in traumatic ulcers, depending on their proportional concentrations [15, 30,31]. HGF maybe a new drug target to treat metabolic disease by inhibiting the inflammation cytokines in adipose tissue in HGF-Tg mice [26]. Anti-inflammatory roles of HGF have been described, including inhibiting pro-and inflammatory cytokines such as TNF-ɑ, IFNγ,TGF-βand iNOS. increasing anti-inflammatory cytokines such as RANTES, IL-4, IL-10 and blocking NF-кB. All these roles could promote the wound healing of oral ulcer [16,32]. We measured the effect of HGF on 40 inflammatory cytokines up to 5 days after the model was constructed. mice. Almost all cytokines had a higher fold-change of fluorescent signal intensity in HGF-Tg group compared with WT mice, except for fractalkine and MIP-1 alpha. The levels of LIX , IL3, eotaxin, eotaxin-2, MIP-1, BLC, RANTES and MCSF were significantly higher in HGF-Tg than in WT after injury. Eotaxin, LIX, Eotaxin-2, MIP-1, BLC, and RANTES are chemokines that are early signals from traumatic tissues to immune cells [30]. This suggests that HGF promotes a stronger signal for the recruitment of inflammatory cells into traumatic tissue and corresponding responses from immune cells [30]. The levels of colonystimulating factors LIX and IL3 were also significantly higher in HGF-Tg, possibly suggesting better tissue recovery after injury [30]. We also observed that almost all the pro-and anti-inflammatory cytokines increased in HGF-Tg mice, although the differences showed no statistical significance. So HGF can contribute to anti-inflammatory effect and wound repair via adjusting cytokine production [10].
The transcription factor nuclear factor kappa-B (NF-кB) is important for inflammatory responses, mediating the expression of cellular signals such as cytokines and growth factors [33]. Several studies have shown that HGF exerts anti-inflammatory action by disrupting NF-кB expression. HGF potently inhibits renal inflammation by preventing p65/NF-кB binding at its acting element [34], or by shutting down NF-кB activation through increasing A20 expression [35], without side effects on normal kidney function [36]. By reducing the activity of NF-кB, increased HGF expression in platelet-rich plasma provides anti-inflammatory function in an articular chondrocyte inflammation model [8]. In this study, there was a significant decrease in connective NF-кB expression in HGF-Tg ulcers on the 5 th day.
However, this effect was not observed in ulcer epithelium; both groups expressed higher epithelial NF-кB. We observed that HGF may decrease connective inflammation of the oral mucosa by reducing connective NF-кB. The mechanism merits further study.

Figures
C57BL/6 (HGF-Tg) mice.   The blood lymphocyte immunophenotypes were labeled with fluorescence antibodies and detected using fluorescence activated cell sorting (FACS). There were no significant differences in terms of T cells, neutrophils, macrophages and the CD8+/CD4+ ratio between the HGF-Tg and WT groups (p>0.05).

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